Determination of blood groups using anti-A and anti-B tsoliclones: Anti-A and anti-B cyclones are designed to determine the human blood groups of the ABO system instead of standard isohem-agglutinating sera.
Determination of blood groups of the ABO system includes the detection of antigens A and B in erythrocytes with standard antibodies and the detection of agglutinins in the serum or plasma of the test blood with standard erythrocytes. Antigens A and B are determined using anti-A and anti-B tsoliclones. In donors, it is mandatory to determine not only antigens in erythrocytes, but also agglutinins in serum (plasma) using standard erythrocytes.
Basic properties of zoliclonal sera.
Anti-A and anti-B cyclones are the product of hybridoma cell lines resulting from the fusion of murine antibody-producing B lymphocytes with murine myeloma cells. Individual hybridoma lines produce homogeneous antibodies of only one class of immunoglobulins, which are completely identical in structure and biological activity. Antibodies produced by cells of one clone (progeny of one cell) are monoclonal.
Cell culture one specificity, originating from one lymphocyte, forms cells of one clone. The main thing is as follows: it is necessary to obtain specific antibodies from one cell and to produce them in unlimited quantities. The difficulty lies in overcoming two tasks: first, normal cells die after several divisions; secondly, only cancer cells are capable of multiplying indefinitely. To solve these two incompatible problems in 1975 G. Kohler and K. Milstein created cell hybrids, or hybridomas. Hybridomas are the fusion of normal lymphocytes in a nutrient medium with myeloma cells. Lymphocytes in this environment do not die, and from the myeloma partner, they get the opportunity to multiply endlessly. Thus, the hybridoma clan multiplies, and as a result of this process, monoclonal antibodies are formed.
Lymphocytes carrying agglutinogen A produce a specific anti-A antibody; lymphocytes carrying agglutinogen B produce a specific anti-B antibody.
Monoclonal anti-A and anti-B antibodies are produced by two different hybridomas. Tsoliklones anti-A and anti-B are diluted ascites fluid of mice-carriers of the corresponding hybridoma, which contains specific immunoglobulins of class M (IgM) directed against group-specific antigens A or B of a person.
Thus, tsoliclones do not contain antibodies of other specificity and therefore do not cause nonspecific poly-agglutination of erythrocytes.
Avidity, that is, the time of the onset of the agglutination reaction and its severity, in anti-A and anti-B tsoliclones is higher than in isohemagglutinating ABO sera, especially in the case of weakly expressed erythrocyte antigens.
Tsolyclones are not cells of human tissues, which excludes the possibility of infection.
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Method for determination blood groups using anti-A and anti-B tsoliclones
Determination of blood groups of the ABO system with tsoliclon reagents is performed in native blood stabilized with the use of preservatives used (glugitsir, citroglucophosphate, heparin, etc.); in blood taken from a finger; in blood taken without preservative. The clearest agglutination reaction is observed when using a high concentration of erythrocytes.
Determination of the blood group is carried out in a room with good lighting at a temperature from + 15 ° to + 25 ° C. Reagents should not be stored open, as antibodies will diminish when they dry. Do not use reagents if they contain insoluble flakes or turbidity. A different labeled (anti-A or anti-B) pipette is used for each reagent. Determination of the blood group of the ABO system is carried out by conventional methods on a white porcelain or any other plate with a wetted surface.
The high activity and avidity of the zoliclon reagents allows the use of one series of anti-A and anti-B reagents. On the plane of the plate or plate, two drops of anti-A and anti-B tsoliclones (0.1 ml) are applied under the appropriate inscriptions: anti-A or anti-B. Next to the drops of antibodies, the test blood is applied in one small drop, approximately 10 times less (0.01 ml). In the case of determining the blood group taken from a finger or taken without a preservative, it is necessary to take a large number of red blood cells, i.e. the first drops from the finger (without strong squeezing) or free red blood cells from the clotted blood sediment.
When determining the blood group, antibodies and blood are mixed with a glass rod or the angle of a glass slide, which are washed and wiped dry before stirring each drop. Observation of the reaction is carried out with light rocking for no more than 2.5 minutes. A positive result in determining the blood group manifests itself as agglutination (gluing) of erythrocytes. At the same time, agglutinates can be seen without any adaptations in the form of small red aggregates that quickly merge and form large flakes.
With a negative reaction to determining the blood group, the drop remains uniformly colored red, no agglutinates are found in it. Agglutination is usually determined within the first 3-5 seconds. Despite this, the results must be monitored for at least 2.5 minutes due to the possibility of a later onset of agglutination with erythrocytes containing weak types of antigens A or B.